This project focuses on molecular properties and regulation of ion channels in T lymphocytes, taking advantage of parallel advances in electrophysiology, molecular biology and video imaging techniques. Our goal is to understand the role of ion channels in the immune response. Using patch-clamp techniques, we have characterized a diverse set of functionally significant ion channels that are differentially expressed depending on the developmental and activation state. Through the proposed experiments, we plan to continue our studies of three main channel types. A voltage-gated K+ channel, Kv1.3, is functionally important in resting T cells. Using site-directed mutants, we will map the channel's inner vestibule with tethered blockers and characterize the action of progesterone. Ca2+- activated K+ channels, encoded by IKCa1 in human T cells and SKCa2 in Jurkat T cells, regulate membrane potentials during [Ca2+]i signaling. IKCa1 is up-regulated in activated T cells and is required for sustained proliferation. We will probe the mechanism of Ca2+ sensing by pre-bound calmodulin. Dominant- negative constructs will be developed to suppress channel expression. Calcium signaling and gene expression depend crucially on Ca2+ release-activated Ca2+ (CRAC) channels that open when intracellular Ca2+ stores are depleted. We will investigate the activation mechanism of this channel, with single-channel resolution, investigate block by polyamines, and use a dominant-negative strategy to test for candidate genes. Finally, using highly specific and potent blockers developed during the previous grant period, we will test for functional roles of K+ channels in [Ca2+]i signaling, cytokine release, cell proliferation, and chemotaxis. Through the proposed experiments we hope to define mechanisms that regulate ion channels and corresponding cell functions that underlie the immune response.